Article pubs.acs.org/jnp
Luteolin Suppresses the Differentiation of THP‑1 Cells through the Inhibition of NOX2 mRNA Expression and the Membrane Translocation of p47phox Junya Makino,† Ryohei Nakanishi,† Tetsuro Kamiya,*,† Hirokazu Hara,† Masayuki Ninomiya,‡ Mamoru Koketsu,‡ and Tetsuo Adachi† †
Laboratory of Clinical Pharmaceutics, Gifu Pharmaceutical University, 1-25-4 Daigaku-nishi, Gifu 501-1196, Japan Department of Chemistry and Biomolecular Science, Faculty of Engineering, Gifu University, 1-1 Yanagido, Gifu 501-1193, Japan
‡
ABSTRACT: Luteolin (1), a natural product occurring in many vegetables and fruits, is known to have several biological activities. Cluster for differentiation (CD) families, such as CD11b, -14, and -36, are expressed during pathological processes of atherosclerosis and are used broadly as markers of monocytic differentiation into macrophages. Herein, it was investigated whether 1 and three other flavonoids [chrysin (2), apigenin (3), and tricetin (4)] blocked 12-O-tetradecanoylphorbol 13-acetate (TPA)-triggered induction of CD families, which were induced through the activation of protein kinase C (PKC), mitogen-activated protein kinase (MEK)/extracellular signal-regulated kinase (ERK), and NADPH oxidase (NOX)-derived reactive oxygen species (ROS). When compared to flavonoids 2−4, 1 blocked TPA-triggered induction of CD families and cell adherence of monocytic THP-1 cells. Luteolin completely blocked intracellular ROS generation, whereas it did not inhibit MEK/ERK phosphorylation. Moreover, pretreatment with 1 suppressed TPA-triggered induction of NOX2 and membrane translocation of p47phox. Overall, it is revealed that 1 suppresses TPA-triggered induction of CD families by the prevention of NOX2 activation.
A
treatment of THP-1 cells with TPA induced the expression of CD11b through ROS generation.12 Flavonoids found commonly in fruits, vegetables, wine, and tea are known to have biological effects, such as antiinflammatory, antioxidative, anticancer, and antiallergy activities.13−17 Therefore, many flavonoids, including luteolin (1), are used broadly as constituents of health food and may prevent vascular injury by ROS.18 Flavonoids can be categorized in various groups according to their different structural types.19 In the present study, the effects of luteolin (1), chrysin (2), apigenin (3), and tricetin (4) on TPA-triggered induction of CD families were examined. It was found that 1 inhibited TPA-triggered induction through the prevention of NOX2 activation and that pretreatment with luteolin resulted in suppression of the monocytic adherence of THP-1 cells.
therosclerosis, which shows the features of an inflammatory disease, is characterized by monocytic differentiation into macrophages and foaming cell formation.1 It is known that the expression of cluster for differentiation (CD) families, such as CD11b, -14, and -36, is a marker of differentiation into macrophages, and the increment of CD family expression is considered to participate in atherosclerosis through the uptake of oxidized low density lipoprotein (LDL) and apoptotic cells into macrophages.2−4 Recently, it has been reported that atherosclerosis is accelerated by the induction of CD families and intracellular reactive oxygen species (ROS) generation.5,6 ROS are known to be generated by activated endothelial cells, macrophages, and leukocytes and have been implicated in various pathological processes, including asthma, diabetes, and atherosclerosis.7,8 Therefore, scavenging ROS may result in suppression of the pathogenesis and development of atherosclerosis. It is well known that leukemic cell lines, such as U937, THP1, and HL-60 cells, can differentiate into macrophages by treatment with various agents, such as 12-O-tetradecanoylphorbol-13-acetate (TPA) and all-trans-retinoic acid (ATRA).9−11 During monocytic differentiation and following phagocytosis, ROS are generated by activated NADPH oxidase (NOX), and the excess production of ROS results in oxidative stress, leading to the dysregulation of signal transduction events and biomolecular injury, all of which contribute to pathological changes in cell and tissue function.7,8 We found previously that © XXXX American Chemical Society and American Society of Pharmacognosy
■
RESULTS AND DISCUSSION Expression of CD11b and CD14 during TPA-Induced THP-1 Differentiation. It is known that TPA induces monocytic differentiation and adhesion.9−11 The expression of CD11b and CD14 is a major marker of monocytic differentiation. Indeed, TPA induced the expression of these Received: March 17, 2013
A
dx.doi.org/10.1021/np400224w | J. Nat. Prod. XXXX, XXX, XXX−XXX
Journal of Natural Products
Article
is the most potent compound found to suppress the induction of CD families among the four flavonoids evaluated. Effect of Luteolin (1) on MEK/ERK Phosphorylation. It is well known that many flavonoids, such as rutin and quercetin, can scavenge various forms of ROS because they have a catechol ring.20 Therefore, it was hypothesized that 1 may have a similar potency to quercetin or rutin in this regard. In a previous report, mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) was found to play a pivotal role in the induction of CD11b during TPAinduced THP-1 differentiation,12 but TPA did not induce the activation of c-jun N-terminal kinase (JNK) and p38 MAPK (data not shown). On the other hand, it has been reported that quercetin inhibits the activity of MEK1 and TPA-induced phosphorylation of ERK,21 so the potential effect of 1 on blocking TPA-triggered MEK/ERK phosphorylation was investigated. However, an inhibitory effect of 1 on MEK and ERK phosphorylation was not observed (Figure 3), suggesting that 1 does not directly inhibit MEK/ERK activation. Effect of Luteolin (1) on NOX2 Activation. It is well recognized that ROS act as signaling molecules involved in various physiological processes, including the monocytic differentiation of phagocytic cells.22−25 NOX2 is known to be most highly expressed in monocytes/macrophages and is essential in innate host defense, both by producing ROS to attack invaders after phagocytosis and by acting as a signal to initiate a number of inflammatory and immunoprotective responses.26,27 During monocytic differentiation and following phagocytosis, ROS are generated by activated NOX2, a multisubunit enzymatic complex comprising two membranebound subunits, gp91 and p22phox. The activation of NOX is
CD families (Figure 1) and morphological changes; floating cells changed to adherent cells (Figure 2). Effect of Flavonoids 1−4 on THP-1 Cell Adhesion and the Induction of CD Families. The effects of flavonoids on TPA-triggered induction of CD families were observed. Pretreatment with 20 μM 1 suppressed TPA-triggered induction of CD families significantly, whereas 0.2 or 2 μM 1 did not (Figure 2A). Accordingly, compounds 1−4 were used at a concentration of 20 μM in subsequent studies. As shown in Figure 2B, 1 suppressed TPA-triggered cell adhesion, whereas 2 and 3 showed a partial suppression, and 4 was inactive (data not shown). Therefore, the effect on the expression of CDs was next compared for these four flavonoids. Pretreatment with 2, 3, or 4 partially or very slightly blocked TPA-triggered induction of CD families, whereas 1 completely blocked this induction (Figure 2C). These results showed that luteolin (1)
Figure 1. TPA induces the expression of CD11b and CD14. THP-1 cells were treated with (closed histogram and column) or without (open histogram and column) 100 nM TPA for 24 h. After the cells had been treated, the expression levels of CD11b and CD14 were measured by flow cytometry (**p < 0.01 vs untreated cells). B
dx.doi.org/10.1021/np400224w | J. Nat. Prod. XXXX, XXX, XXX−XXX
Journal of Natural Products
Article
Figure 2. Effect of flavonoids on the TPA-triggered induction of CD families and cell adhesion. (A) THP-1 cells were pretreated with 0.2, 2, or 20 μM luteolin (1) for 1 h and then treated with (+) or without (−) 100 nM TPA for 24 h. (B) Cells were pretreated with or without 20 μM 1 for 1 h and then treated with or without 100 nM TPA for 24 h. After the cells had been treated, cell adhesion was detected using a microscope. (C) Cells were pretreated with 20 μM of the flavonoid luteolin (1), chrysin (2), apigenin (3), or tricetin (4) for 1 h and then treated with (+) or without (−) 100 nM TPA for 24 h. After the cells had been treated, RT-PCR was carried out. Data were normalized using 18s rRNA levels (*p < 0.05, **p < 0.01 vs untreated cells, #p < 0.05, ##p < 0.01 vs TPA-treated cells).
TPA-triggered membrane translocation of p47phox was investigated. As shown in Figure 4A, 1 completely blocked TPAinduced ROS generation. Additionally, luteolin also blocked both TPA-triggered induction of NOX2 and membrane translocation of p47phox (Figure 4B and C). From these results, it was considered that 1 blocks TPA-induced ROS generation through the inhibition of NOX2 activation. On the other hand, tricetin (4) did not suppress the induction of NOX2 mRNA and membrane translocation of p47phox (Figure 4B and C), suggesting that there are some differences in the site of action between 1 and 4. In this study, luteolin (1), with a catechol ring, was considered as the most likely compound to inhibit TPAtriggered induction of CDs. However, it was confirmed that 4
Figure 3. Effect of luteolin (1) on TPA-triggered MEK/ERK phosphorylation. THP-1 cells were pretreated with (+) or without (−) 20 μM 1 for 1 h and then treated with (+) or without (−) 100 nM TPA for 15 min. After the cells had been treated, phosphorylated and total MEK or ERK levels were determined by Western blotting.
regulated by cytoplasmic subunits such as p47phox, p67phox, and the small G protein Rac1.28−31 Therefore, the effect of 1 on C
dx.doi.org/10.1021/np400224w | J. Nat. Prod. XXXX, XXX, XXX−XXX
Journal of Natural Products
Article
Figure 5. Hypothesis of the site of action of luteolin (1). CD families are induced during monocytic differentiation through protein kinase C (PKC), MEK/ERK, and NOX2-derived ROS. 1 has the potent activities of the blockage of NOX2 mRNA expression and membrane translocation of p47phox.
Overall, luteolin (1) has various effects as an antioxidant, and optimal intake of this compound where present in vegetables and fruits may contribute to the prevention of atherosclerosis.
■
Figure 4. Effect of luteolin (1) on TPA-triggered ROS generation and NOX2 activation. (A) THP-1 cells were pretreated with (+) or without (−) 20 μM 1 for 1 h and then treated with (+) or without (−) 100 nM TPA for 24 h. After the cells had been treated, intracellular ROS accumulation was measured by flow cytometry (**p < 0.01 vs untreated cells, ##p < 0.01 vs TPA-treated cells). (B) The cells were pretreated with (+) or without (−) 20 μM 1 or 4 for 1 h and then treated with (+) or without (−) 100 nM TPA for 24 h. After the cells had been treated, NOX2 expression was measured by RT-PCR. RTPCR data were normalized using 18s rRNA (*p < 0.05, **p < 0.01 vs untreated cells, ##p < 0.01 vs TPA-treated cells, N.S., not significant vs TPA-treated cells). (C) Cells were pretreated with (+) or without (−) 20 μM 1 or 4 for 1 h and then treated with (+) or without (−) 100 nM TPA for 12 h. After the cells had been treated, the membrane translocation of p47phox was determined by Western blotting.
EXPERIMENTAL SECTION
Reagents. 12-O-Tetradecanoylphorbol 13-acetate (TPA) was purchased from Sigma-Aldrich Co. (St Louis, MO, USA). 5-(and-6)Carboxy-2′,7′-dichlorodihydrofluorescein diacetate (carboxyH2DCFDA) was purchased from Molecular Probes (Eugene, OR, USA). Anti-MEK rabbit polyclonal antibody, anti-phospho-MEK rabbit polyclonal antibody, anti-ERK rabbit monoclonal antibody, and anti-phospho-ERK mouse monoclonal antibody were purchased from Cell Signaling Technology (Danvers, MA, USA). Anti-p47phox rabbit polyclonal antibody and anti-actin mouse monoclonal antibody were purchased from Millipore Co. (Billerica, MA, USA). Biotinconjugated goat anti-rabbit and anti-mouse IgG (H+L) were purchased from Invitrogen (Carlsbad, CA, USA). Luteolin (1) (>98.0%) and chrysin (2) (>98.0%) were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan) and CosmoBio Co., Ltd. (Tokyo, Japan), respectively. Apigenin (3) and tricetin (4) were synthesized by the methods described in a previous report.34 Their purities were assessed as >95% using analytical HPLC. Cell Culture. THP-1 cells were cultured in RPMI1640 medium containing 10% (v/v) heat-inactivated fetal calf serum, 100 U/mL penicillin, and 100 μg/mL streptomycin at 37 °C in a humidified 5% CO2 incubator. For the differentiation of THP-1 cells, they were seeded at 2 × 106 cells in 3.5 cm dishes, and 100 nM TPA was added. After differentiation, the cells were scraped and washed with cold phosphate-buffered saline (PBS) followed by the extraction of total RNA, flow cytometry analysis, and Western blotting. Cell Adhesion. THP-1 cells were treated with TPA for 24 h at a concentration of 2 × 106 cells in 3.5 cm dishes. After differentiation, the cells were washed three times with PBS. Cell adhesion was observed under a microscope. Reverse-Transcriptional Polymerase Chain Reaction (RTPCR) Analysis. After THP-1 cells had been treated with the reagents, they were lysed in 1 mL of TRIzol reagent (Invitrogen, Carlsbad, CA, USA). The cDNA was prepared, and RT-PCR was performed by the method described in a previous report,35 with minor modifications. The primer sequences used in this study were as follows: CD 11b, sense 5′-CCC CCA GGT CAC CTT CTC CG-3′; antisense 5′-GCT CTG TCG GGA AGG AGC CG-3′ [525 base pair (bp)]; CD14, sense 5′-CGA GGA CCT AAA GAT AAC CGG C-3′; antisense 5′GTT GCA GCT GAG ATC GAG CAC-3′ (510 bp); CD36, sense 5′TGC CTC TCC AGT TGA AAA CCC-3′; antisense 5′-GCA ACA
did not block TPA-triggered induction of CDs despite the fact that 4 has one more hydroxy group in ring B than 1. It has been reported that 4 induces apoptosis through glutathione depletion and ROS generation in human liver cancer cells.32 It is thought that 1 acts as an antioxidant, whereas 4 acts as a pro-oxidant and induces the expression of CD11b in THP-1 cells (Figure 2C). On the other hand, it has also been reported that luteolin (1) and nobiletin, a methoxylated flavonoid, show differences in their absorption and accumulation in rat tissue.33 Although it is not known exactly which flavonoids can be taken up by THP-1 cells efficiently, the differences in permeability between luteolin (1) and flavonoids 2−4 might be involved in their differential inhibitory effects against TPA-triggered CD induction. However, additional studies will be necessary to determine the different mechanisms of 1 and 2−4. In conclusion, a new site of action of 1 as an antioxidant may be postulated (Figure 5). It was revealed that 1 did not suppress MEK and ERK phosphorylation, but blocked TPAtriggered induction of CD families and membrane translocation of p47phox. Moreover, 1 also inhibited TPA-induced adhesion of THP-1 cells through the suppression of these processes. D
dx.doi.org/10.1021/np400224w | J. Nat. Prod. XXXX, XXX, XXX−XXX
Journal of Natural Products
Article
AAC ATC ACC ACA CCA-3′ (484 bp); NOX2, sense 5′-GGA GTT TCA AGA TGC GTG GAA CTA-3′; antisense 5′-GCC AGA CTC AGA GTT GGA GAT GCT-3′ (550 bp); 18s rRNA (rRNA), sense 5′-CGG CTA CCA CAT CCA AGG AA-3′, antisense 5′-GCT GGA ATT ACC GCG GCT-3′ (187 bp). These PCR products were loaded on a 2% (w/v) agarose gel for electrophoresis, and densitometric analysis of the PCR products was performed with Multi Gauge V3.0 (Fuji Film, Tokyo, Japan). Western Blotting. Whole cell extracts were prepared in lysis buffer, as described previously.12,36 For phosphorylated protein detection, the cells were scraped and lysed in 200 μL of lysis buffer A [20 mM Tris-HCl, pH 7.4, containing 1 mM EDTA, 1 mM EGTA, 10 mM NaF, 1 mM Na3VO4, 20 mM β-glycerophosphate, 1 mM phenylmethylsulfonyl fluoride (PMSF), 1 mM dithiothreitol (DTT), 2 μg/mL leupeptin, and 1% Triton X-100]. For the preparation of a membrane fraction, the cells were scraped and lysed in 100 μL of lysis buffer B (20 mM Tris-HCl, pH 7.5, 2 mM EDTA, 10 mM EGTA, 1 mM DTT, 100 μg/mL digitonin, 10 mM NaF, 1 mM Na3VO4, 20 mM β-glycerophosphate, 1 mM PMSF). After centrifugation at 12000g for 15 min, soluble fractions were removed and the remaining pellets were treated for 15 min on ice with 100 μL of lysis buffer B supplemented with 1% Triton X-100. Extracts containing 20 μg of protein were separated by SDS-PAGE on a 12% (w/v) polyacrylamide gel followed by transferring electrophoretically onto PVDF membranes. Subsequently, the membranes were incubated with the respective specific primary antibodies (1:1000). The blots were incubated with biotinconjugated goat anti-rabbit or anti-mouse antibody (1:1000) followed by incubating with ABC reagents (Vector Laboratories, Inc., Burlingame, CA, USA) (1:5000). Finally, the bands were detected using SuperSignal West Pico (Thermo Scientific, Rockford, IL, USA) and imaged using an LAS-3000 UV mini apparatus (Fuji Film, Tokyo, Japan). Analysis of CD11b and CD14 Expression. Differentiation of THP-1 cells was assessed by analysis of the expression of CD11b and CD14 surface markers. After the cells had been treated, they were scraped followed by washing with ice-cold PBS and centrifugation. The pellets were stained with CD11b-PE and CD14-FITC antibodies in PBS containing 1% paraformaldehyde (PFA) in a 5% CO2 incubator for 20 min. After incubation, the cells were washed with ice-cold PBS three times and resuspended in 1% PFA−PBS. Fluorescent intensities were analyzed by FACS scan (BD Biosciences, San Jose, CA, USA). Analysis was performed using BD CellQuest Pro Software. Measurement of Intracellular ROS Accumulation. Intracellular ROS accumulation was measured according to the method described previously,12,36 with minor modifications. Briefly, the cells were suspended followed by staining with 10 μM carboxy-H2DCFDA in PBS containing 1% PFA for 20 min in a 5% CO2 incubator. After incubation, the cells were washed with ice-cold PBS three times and resuspended in 1% PFA−PBS. Fluorescent intensities of DCF were analyzed by FACS scan (BD Biosciences, San Jose, CA, USA). Analysis was performed using BD CellQuest Pro Software. Statistical Analysis. Data are expressed as the means ± SD of three independent experiments. Statistical evaluation of the data was performed using ANOVA followed by a posthoc Bonferroni test. A p value of less than 0.05 is considered significant.
■
encouragement of young scientists from Gifu Pharmaceutical University (T.K.).
■
REFERENCES
(1) Ross, R. N. Engl. J. Med. 1999, 340, 115−126. (2) Rahaman, S. O.; Lennon, D. J.; Febbraio, M.; Podrez, E. A.; Hazen, S. L.; Silverstain, R. L. Cell Metab. 2006, 4, 211−221. (3) Smirnoval, I. V.; Kajstura, M.; Sawamura, T.; Goligorsky, M. S. Am. J. Physiol. Heart Circ. Physiol. 2004, 287, 782−790. (4) Gu, B. J.; Saunders, B. M.; Petrou, S.; Wiley, J. S. J. Immunol. 2011, 187, 2365−2375. (5) Park, Y. M.; Febbraio, M.; Silverstein, R. L. J. Clin. Invest. 2009, 119, 136−145. (6) Park, J. G.; Oh, G. T. BMB Rep. 2011, 44, 497−505. (7) Vachier, I.; Chanez, P.; Le, D. C.; Damon, M.; Descomps, B.; Godard, P. Eur. Respir. J. 1994, 7, 1585−1592. (8) Wright, E., Jr.; Scism-Bacon, J. L.; Glass, L. C. Int. J. Clin. Pract. 2006, 60, 308−314. (9) Miranda, M. B.; McGuire, T. F.; Johnson, D. E. Leukemia 2002, 16, 683−692. (10) Pathak, M. K.; Hu, X.; Yi, T. Leukemia 2002, 16, 2285−2291. (11) Barbieri, S. S.; Eligini, S.; Brambilla, M.; Tremoli, E.; Colli, S. Cardiovasc. Res. 2003, 60, 187−197. (12) Makino, J.; Kamiya, T.; Hara, H.; Adachi, T. Free Radical Res. 2012, 46, 637−644. (13) Rusak, G.; Gutzeit, H. O.; Ludwig-Muller, J. Nutr. Res. (N.Y.) 2005, 25, 143−155. (14) Yi, L.; Chen, C. Y.; Jin, X.; Zhang, T.; Zhou, Y.; Zhang, Q. Y.; Zhu, J. D.; Mi, M. T. Biochemie 2012, 94, 2035−2044. (15) Jung, H. A.; Jin, S. E.; Min, B. S.; Kim, B. W.; Choi, J. S. Food Chem. Toxicol. 2012, 50, 2171−2179. (16) Jin, T. H.; Ock, J. P.; Yun, K. L.; Mi, J. S.; Haeng, J. H.; Myung, S. K.; Joo, H. H.; Dae, Y. K. Int. J. Mol. Med. 2011, 28, 25−31. (17) Seelinger, G.; Merfort, I.; Schempp, C. M. Planta Med. 2008, 74, 1667−1677. (18) Woodman, O. L.; Chan, E. C. Clin. Exp. Pharmacol. Physiol. 2004, 31, 786−790. (19) Guerrero, L.; Castillo, J.; Quinones, M.; Garcia-Vallve, S.; Arola, L.; Pujadas, G.; Muquerza, B. PLoS One 2012, 7, 49493. (20) Kessler, M.; Ubeaud, G.; Jung, L. J. Pharm. Pharmacol. 2003, 55, 131−142. (21) Ki, W. L.; Nam, J. K.; Yong-Seok, H.; Evgeny, A. R.; Angelo, P.; Mun, K H.; Bowden, G. T.; Ann, M. B.; Hyong, J. L.; Zigang, D. Cancer Res. 2008, 68, 946−955. (22) Droge, W. Physiol. Rev. 2002, 82, 47−95. (23) Finkel, T. Curr. Opin. Cell Biol. 2003, 15, 247−254. (24) Nathan, C. J. Clin. Invest. 2003, 111, 769−778. (25) Kiningham, K. K.; Cardozo, Z. A.; Cook, C.; Cole, M. P.; Stewart, J. C.; Tassone, M.; Coleman, M. C.; Spitz, D. R. Free Radical Biol. Med. 2008, 44, 1610−1616. (26) Rada, B.; Hably, C.; Meczner, A.; Timár, C.; Lakatos, G.; Enyedi, P.; Ligeti, E. Semin. Immunopathol. 2008, 30, 237−253. (27) Brown, D. I.; Griendling, K. K. Free Radical Biol. Med. 2009, 47, 1239−1253. (28) Babior, B. M. Blood 1991, 93, 1464−1476. (29) Ichikawa, H.; Kokura, S.; Aw, T. Y. J. Vasc. Res. 2004, 41, 432− 444. (30) Bedard, K.; Krause, K. H. Physiol. Rev. 2007, 87, 245−313. (31) Abo, A.; Pick, E.; Hall, A.; Totty, N.; Teahan, C. G.; Segal, A. W. Nature 1991, 353, 668−670. (32) Hsu, Y. L.; Hou, M. F.; Tsai, E. M.; Kou, P. L. J. Agric. Food Chem. 2010, 58, 12547−12556. (33) Murakami, A.; Koshimizu, K.; Ohigashi, H.; Kuwahara, S.; Kuki, W.; Takanishi, Y.; Hosotani, K.; Kawahara, S.; Matsuoka, Y. Biofactors 2002, 16, 73−82. (34) Ninomiya, M.; Tanaka, K.; Tsuchida, Y.; Muto, Y.; Koketsu, M.; Watanabe, K. J. Med. Chem. 2011, 54, 1529−1536. (35) Yamamoto, M.; Hara, H.; Adachi, T. Atherosclerosis 2002, 163, 223−228.
AUTHOR INFORMATION
Corresponding Author
*Tel: +81 58 230 8100. Fax: +81 58 230 8105. E-mail:
[email protected]. Notes
The authors declare no competing financial interest.
■
ACKNOWLEDGMENTS This study was supported in part by a Grant-in-Aid for Scientific Research from the Japan Society for the Promotion for Science (T.K.: No. 2379019) and a grant for the E
dx.doi.org/10.1021/np400224w | J. Nat. Prod. XXXX, XXX, XXX−XXX
Journal of Natural Products
Article
(36) Kamiya, T.; Makino, J.; Hara, H.; Inagaki, N.; Adachi, T. J. Cell Biochem. 2011, 112, 244−255.
F
dx.doi.org/10.1021/np400224w | J. Nat. Prod. XXXX, XXX, XXX−XXX
